Central to human Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a large and spacious phagolysosome-like parasitophorous vacuole (PV). Recruitment of membrane required for PV biogenesis is a complex process modulated by host and bacterial factors. We have shown that the PV membrane is cholesterol-rich and that pharmacologic inhibition of host cholesterol metabolism negatively impacts PV generation and pathogen replication. Cholesterol is a critical component of mammalian membranes where it provides structural stability, signaling platforms called lipid rafts, and serves as a precursor of secondary messenger molecules. To better understand the role of cholesterol in Coxiella pathogenesis, and to circumvent potential pleiotropic effects of cholesterol metabolism inhibitors, we developed a cholesterol-free cell system using DHCR24-/- mouse embryonic fibroblasts (MEFs) that lack the mammalian &#916;24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis. Membranes of these cells accumulate desmosterol-a sterol unable to form lipid rafts-instead of cholesterol. The ability of Coxiella to colonize DHCR24-/- MEFs was investigated along with colonization by Chlamydia trachomatis and Salmonella typhimurium as control organisms. Uptake of Salmonella and Chlamydia was unaltered in DHCR24-/- MEFs. Moreover, secretion of Salmonella type III effectors, essential for host invasion, was not affected in the absence of cholesterol. In contrast, Coxiella was internalized less efficiently in DHCR24-/- MEFs, suggesting a role for cholesterol-rich lipids rafts in Coxiella host cell entry. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, in DHCR24-/- MEFs, the Coxiella PV lacked the typical multilamellar membranes found in wild type cells, suggesting cholesterol plays a role in vesicle trafficking to the PV. These data indicate cholesterol is not essential for invasion and intracellular replication by Salmonella and Chlamydia, but may play a role in Coxiella-host cell interactions. We have identified 40 Coxiella Dot/Icm Type IV secretion system (T4SS) substrates that represent a treasure trove of potential virulence factors. Elucidation of their cellular activities and targets will provide needed information on the Coxiella/host relationship. Coxiella Dot/Icm substrates were initially identified using Legionella as surrogate host. However, by using new Coxiella genetic transformation methods developed in our laboratory, we have confirmed Dot/Icm dependent secretion of effectors by Coxiella. An interesting subset of six effectors is encoded by the Coxiella cryptic QpH1 plasmid. When ectopically expressed in HeLa cells, plasmid effectors traffic to different subcellular sites, including autophagosomes, ubiquitin-rich compartments, and the endoplasmic reticulum Collectively, these results suggest Coxiella plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, thereby providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen. Ectopic expression in mammalian cells of chromosomally encoded effectors fused to fluorescent proteins also reveals a variety of subcellular localizations including microtubules and the Coxiella PV membrane. Yeast two-hybrid analysis identified potential eucaryotic binding partners for six Coxiella Dot/Icm substrates. These preliminary results now set the stage for defining effector function. Indeed, we currently have Coxiella transformants expressing T4SS effectors fused to epitope tags for overexpression by Coxiella. The subcellular trafficking of tagged effectors will provide important clues concerning function. We have also generated a Coxiella strain with the icmD gene inactivated with the Himar1 transposon (Tn). This strain was recently used to define the requirements of type IV secretion during Coxiella infection of human macrophages. An intracellular biphasic developmental cycle whereby highly resistant small cell variant (SCV) morphological forms are generated from large cell variant (LCV) morphological forms is considered fundamental to Coxiella virulence. Previous work from our lab revealed that the LCV is the replicative form of Coxiella, and that SCV and LCV are compositionally and antigenically different. Further molecular and biochemical analyses of SCV and LCV morphogenesis is necessary to better understand the physiological relevance of Coxiella biphasic development. However, intracellular growth of Coxiella imposes considerable experimental constraints. Therefore, we investigated development in our new host cell-free growth medium, Acidified Cysteine Citrate Medium (ACCM). SCV to LCV transitions in ACCM are indistinguishable from Coxiella propagated in vivo. The fidelity of Coxiella morphogenesis in ACCM now provides ample pure cell populations for biochemical studies, ultrastructural analyses, and phenotyping.